Trends of Microdiversity Reveal Depth-Dependent Evolutionary Strategies of Viruses in the Mediterranean

The evolutionary interactions between viruses and their prokaryotic hosts remain a little-known aspect of microbial evolution. Most studies on this topic were carried out in pure cultures that challenge one virus with one bacterial clone at a time, which is very removed from real-life situations. Few studies have addressed trends of microdiversity in marine viral communities throughout depth gradients. We analyzed metagenomes from both the cellular and viral fractions of Mediterranean seawater samples spanning the epipelagic to the bathypelagic zones at depths of 15, 45, 60, and 2,000 m during the summer stratification of the water column. We evaluated microdiversity patterns by measuring the accumulation of synonymous and nonsynonymous mutations in viral genes. Our results demonstrated clear depth-dependent trends in the frequency of polymorphic sites and nonsynonymous mutations among genes encoding metabolic, structural, and replication proteins. These differences were linked to changes in energy availability, host and viral densities, and the proportions of actively replicating viruses. We propose the hypothesis that in the energy-rich, high-host-density, euphotic depths, selection acts to favor diversity of the host recognition machinery to increase host range, while in energy-depleted aphotic waters, selection acts on viral replication fitness, enhancing diversity in auxiliary metabolic genes. IMPORTANCE Viruses are extremely abundant and diverse biological entities that contribute to the functioning of marine ecosystems. Despite their recognized importance, few studies have addressed trends of mutation accumulation in marine viral communities across depth gradients. By investigating these trends, we show that mutation frequencies differ among viral genes according to their molecular functions, with the highest microdiversity occurring among proteins related to host metabolism, followed by structural proteins and, lastly, genome replication proteins. This is in agreement with evolutionary theory that postulates that housekeeping genes are under strong purifying selection. We also observed a positive association between depth and microdiversity. One exception to this trend was the host recognition proteins from the deep chlorophyll maximum, which displayed strikingly high microdiversity, which we hypothesize to be associated with intraspecies competition for hosts. Finally, our data allowed us to propose a theoretical model for viral micro-diversity across the depth gradient. These discoveries are of special relevance because many of the viral genomic sequences discovered here were predicted to infect some of the most abundant bacteria in marine ecosystems, such as Candidatus Pelagibacter, Puniceispirillum, and Prochlorococcus.